Supplementary Materialsoncotarget-10-5229-s001. extra to those resulting from standard treatments (cisplatin +/C irradiation) alone. = 3). (B) Cell viability is compared to untreated controls following 72 (cell lines = 3C7) or 96 (patient-derived primary tumor cells = 6) hours exposure to quinacrine alone (black line) at increasing concentrations and also in the presence of cisplatin (red line). IC50 values are highlighted by vertical dotted lines color matched; responses fitted to a five-parameter logistic PD184352 equation. (C) Viability of cells exposed to 1 M quinacrine compared to untreated controls. (D) Summary of IC50 values (shaded columns) and inverse log of the IC50 values (pIC50) of quinacrine and standard error of the mean (SEM) in each cell line, with and without the addition of cisplatin. To expand this finding, a larger panel of HNSCC cell lines (CAL27, SCC040, FaDu, SCC47 and VU147) was exposed to a range of quinacrine concentrations. The resulting concentration-response curves illustrate that quinacrine effectively inhibits cell viability in a concentration dependent manner (Figure 1B) with IC50 values for cell lines tested ranging from 0.63 to 1 1.21 M (Figure 1B and ?and1D),1D), which is comfortably within clinically achievable concentrations [19C21]. These data indicated that quinacrine was a viable candidate for further development. Quinacrine increases the efficacy of cisplatin HNSCC cell lines demonstrated extra suppression of cell viability when quinacrine was coupled with cisplatin (cell range IC50: 2, 3 or 10 M), in comparison to quinacrine only (Shape 1B). The mix of quinacrine and regular of treatment cisplatin was looked into further (Shape 2A). To show a concentration-dependent decrease in viability, our HNSCC cell lines had been exposed to raising concentrations of cisplatin, with and without the addition of quinacrine at 0.4, 1.5 and 3 M. Quinacrine improved the power of cisplatin to suppress cell viability in every cell lines. This decrease was more apparent at lower concentrations of cisplatin, since cisplatin concentrations of 0.1 mM (10C4 M) or above led to dramatic suppression of viability of most cell lines, in a way that additional suppression with the addition of quinacrine had not been possible. For instance, when treated having a cisplatin focus of 0.3 M (3 10C7 M) alone, SCC040 showed cell viability suppression of 12%, in comparison to suppression of 17%, 48% and 79% following a addition of 0.4, 1.5 and 3 M quinacrine to 0.3 M cisplatin, PD184352 respectively. Compared, at a cisplatin focus of 0.1 mM (10C4M), cell viability was reduced by Ntrk3 cisplatin alone by 90%, PD184352 with just marginal additional suppression by increasing dosages of quinacrine. Open up in another window Shape 2 (A) Focus response curves of cell lines (= 3C4) and patient-derived major tumor cells (= 6) to raising concentrations of cisplatin (reddish colored range) with the addition of 0.4 (black line), 1.5 (green line), 3 (purple line) or 6 M (grey line) quinacrine. Vertical lines highlight IC50 values color matched; responses fitted to a five-parameter logistic equation. (B) Fraction affected vs Combination Index (Fa-CI) plot for each cell line, produced using CompuSyn. Concentrations range from 1/32x C 2x IC50 for quinacrine (Q), and from 1/8x C 8x IC50 for cisplatin (C), maintaining PD184352 a ratio of 1 1:4 Q:C. Data points below 1 (dotted line) represent synergy (= 3). (C) Dose reduction index (DRI) table for CAL27, SCC040 and SCC47 cell lines (= 3). Green indicates DRI values 1 (favorable reduction); orange indicates DRI values 1 (less favorable reduction). Quinacrine displays synergy with cisplatin To confirm the above findings and to assess potential synergy of quinacrine when combined with cisplatin, Chou-Talalay analysis was undertaken [22]. Synergy was observed at lower concentrations of quinacrine and cisplatin, as demonstrated by a combination index (CI) number less than 1 (Figure.